Understanding the mechanism of DNA replication and chromatin inheritance in human cells is a fundamental problem in biology and has direct relevance to cancer research. Alterations to the normal pattern of inheritance can lead to human disease, including cancer. Our studies focus on three specific areas of research that cover different aspects of how a cell inherits chromosomes during cell division. The first area involves the PCNA clamp loader protein, Replication Factor C (RFC) and RFC-related proteins. Biochemical studies are designed to determine the functions of the RFC-like proteins and determine how interact with PCNA-like checkpoint proteins. PCNA is also required for DNA replication dependent chromatin inheritance and inheritance of epigenetically determined states of gene expression. We will investigate the role of PCNA and an associated protein called Chromatin Assembly Factor-1 (CAF-1) in these processes. The identification of other proteins that cooperate with PCNA and CAF-1 will contribute to an understanding of how allele-specific inactivation of gene expression occurs, a process that often leads to loss of tumor suppressor gene function during progression toward cancer. Finally, we will continue studies on the role of human ORC in establishing the sites in chromosomes for nitiation of DNA replication. ORC cooperates with other initiation proteins to form a pre-replication complex in cells and this complex renders chromosomes competent for initiation of DNA replication. We intend to identify ORC binding proteins and characterize their role in chromosome inheritance. These studies on chromosome inheritance are directly relevant to the overall goals of the Program Project.